Over the past decades there has been a keen interest in engineering cellular metabolism for the production of “green” chemicals that could ween us off of our reliance on petrochemicals. One method is to perform a desired biochemical conversions with purified enzymes or cell extracts. Cell free metabolic systems have many advantages over in vivo efforts such as continuous product production, ease of product removal, near 100% yields, and no cell toxicity issues.
Building cell free pathways that can economically sustain high flux for long periods of time without the metabolic regulatory systems that exist in cells requires new design principles, a field referred to as “synthetic biochemistry”. A key consideration in synthetic biochemistry system design is the generation, regulation and recycling of high energy cofactors such as ATP, NADH and NADPH. Generally high energy cofactors are generated in a catabolic or breakdown phase (e.g. glycolysis), then utilized and regenerated in an anabolic or build phase where the desired chemicals are constructed. The simplest way to design synthetic biochemistry systems is to demand perfect stoichiometry, so that if two ATP are generated in the breakdown phase, then two ATP are utilized in the build phase. Stoichiometric systems can allow flux through the pathway for a period of time, but the second law of thermodynamics dictates that they will eventually wind down as ATP is hydrolyzed or NADH is oxidized by undesired side reactions.